WFA alters Filamin A organization but not its expression.

<p>(A) RbCF8 cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.g001" target="_blank">Fig 1</a> in presence or absence of WFA or Epox for 30 min. Control and WFA-treated cells were fixed and stained with anti-pSer38vim (green) and anti-FLNA (red) antibodies. Scale bar = 35 μm at 30X magnification. (B) Control, WFA and Epox-treated cells cultured with drug at time of plating for 1 h (Right Panels) or cells allowed to attach for 30 min and then treated with WFA or Epox for additional 30 min (Left Panels). Equal amount of soluble proteins were fractionated by SDS-PAGE and blots were probed with anti-FLNA antibody. β-actin antibody was used as loading control. (C) Serum-starved cells were trypsinized and replated in 10% serum-containing medium in presence or absence of WFA for 1 h or allowed to attach for 30 min and then treated with WFA for additional 30 min. Equal amount of soluble proteins were fractionated by SDS-PAGE and blots were probed with anti-FLNA and anti-gigax antibodies.</p

Mature focal adhesions require coalescence with vimentin and develop in a cell-passage dependent manner.

<p>(A) RbCF2 and RbCF8 cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.g001" target="_blank">Fig 1</a>, plated for 30 min followed by treatment in presence or absence of WFA (1 μM) for 1 h. Cells were fixed and stained with paxillin (green), vimentin (red), and DAPI (blue). Scale bar = 35 μm. (B) Peripheral FA mean areas from RbCF2 (black columns) and RbCF8 (grey columns) cells were measured and represented in arbitrary units. (*P <0.0001; **P <0.0001).</p

Distribution of vimentin IFs controls filament rearrangement during cell spreading.

<p>RbCF2 and RbCF8 cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.g001" target="_blank">Fig 1</a> and plated for 30 min followed by treatment in presence or absence of WFA (1 μM) for different time points. Cells were fixed and stained (A) vimentin (green) and DAPI (blue) or (B-C) with pSer38vim (pSer38vim, green) and DAPI (blue). Scale bar = 35 μm. Insert panels in panels B and C represent 60X magnification of selected areas. Scale bar = 20 μm.</p

ERK Activation is cell passage dependent.

<p>(A) Western blot analysis of RbCF2, RbCF5 and RbCF8 cells. Cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.g001" target="_blank">Fig 1</a>, plated for 30 min and treated in presence or absence of WFA and Epox for 30 min. Equal amount of soluble proteins were fractionated by SDS-PAGE and blots were probed with anti-pERK1/2 and β-actin antibodies. (B) RbCF8 cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.g001" target="_blank">Fig 1</a>, plated for 30 min and treated in presence or absence of WFA for 30 min. Cells were fixed and stained for pSer38vim (green) and pERK1/2 (red). N = Nucleus. Scale bar = 35 μm at 30X magnification. (C) Western blot analysis of RbCF8 cells. Serum-starved cells were trypsinized, plated for 30 min and then treated with WFA (250 nM and 1 μM) for indicated times. Equal amount of nuclear and cytoplasmic extracts were fractionated by SDS-PAGE and blots were probed with anti-pERK1/2 antibody. Blots were then stripped and reprobed with anti-ERK1/2 antibody as loading control. (D) Immunoprecipitation analysis of RbCF8 cells. Cells were serum-starved 48 h, trypsinized and then replated in presence or absence of different doses of WFA. Equal amount of soluble cell lysates were immunoprecipitated with anti-pERK1/2 antibody and then probed with anti-pSer38vim antibody. Filamin A (FlnA) was used as loading control.</p

WFA controls gel contraction properties in a cell-passage dependent manner.

<p>(A) Representative images of RbCF2 and RbCF8 cells embedded in collagen type I gels and treated with TGF-β1 (2 ng/ml) for 2 days in presence or absence of different doses of WFA. Gels were then fixed and images were captured at 2X magnification. (B) Quantification of gel contractile activity. Images from (A) were used to arbitrarily measure the collagen area in each well using ImageJ program (n = 8 wells/treatment). (C) Immunohistochemistry of RbCF2 and RbCF8 cells cultured on slides and stained with α-SMA (red), anti-vimentin (green) and DAPI (blue). Scale bar = 215 μm at 10X magnification. (D) Western blot analysis of RbCF2 and RbCF8 cells for α-SMA expression. Cells were trypsinized and plated for 30 min and then treated with 1 μM WFA for 30 min or treated with WFA at time of plating for 1 h. Blots were probed with anti-α-SMA and FLNA antibodies used as loading control.</p

Vimentin phosphorylation at other serine residues.

<p>RbCF2 and RbCF8 cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.g001" target="_blank">Fig 1</a>, plated for 30 min followed by treatment in presence or absence of WFA (1 μM) for 1 h. (A) Equal amount of soluble proteins were fractionated by SDS-PAGE and blots were probed with anti-S82vimentin (clone MO82), anti-S71vimentin (clone TM71) and anti-S72vimentin (clone TM72), respectively. Blots were then stripped and re-probed for loading controls. (B) Densitometric quantification of repeat experiments (n = 3; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.s002" target="_blank">S2</a> Figs) of S82Vim, S72Vim and S71Vim normalized to loading controls, using NIH ImageJ software.</p

WFA blocks activation of the ERK1/2 pathway <i>in vivo</i> by targeting pSer38vim expression.

<p>Anesthetized mice were subjected to bilateral corneal alkali injury. Injured mice were treated with DMSO (Vehicle) or WFA (2 mg/kg solubilized in DMSO) on the day of injury and every subsequent day by intraperitoneal injection for 14 days. (A) Immunohistochemistry analysis of WT corneal sections stained with pSer38vim antibody (red) and DAPI (blue). Epi: Epithelium; Str: Stroma; End: Endothelium. Enlarged panels: 60X magnification. (B) Immunohistochemistry analysis of WT and Vim KO samples stained with anti-pERK1/2 antibody (Green) and DAPI (blue). Enlarged panel: 60X magnification of WT-Vehicle sample, showing nuclear expression pERK1/2 (Green). Pointed-ended arrows indicate nuclear localization of pERK1/2 in epithelial and stromal cells of WT-Vehicle samples. Square-ended arrow indicates cytoplasmic localization of pERK1/2 in the basal layer of the epithelium of WT-WFA samples. Arrowhead indicates partial cytoplasmic localization of pERK1/2 in stromal cells of Vim KO-Vehicle samples. (C) Western blot analysis of epithelial and stromal extracts from corneas of mice 14 d post-injury. Equal amount of total protein extracts were fractionated by SDS-PAGE and blots were probed with anti-pERK1/2 antibody. β-tubulin was used as loading control. (D) Western blot analysis of corneal extracts from uninjured WT mice treated for 7 d with WFA (2 mg/kg/d) or vehicle (DMSO). Total protein extracts were fractionated by SDS-PAGE and blots were probed with anti-vimentin, and anti-pERK1/2 antibodies. β-actin antibody was used as loading control.</p

Hyperphosphorylation of soluble pSer38vim by WFA is cell passage dependent.

<p>(A) RbCF2 and RbCF8 cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.g001" target="_blank">Fig 1</a> and plated for 1 h. Equal amounts of soluble and insoluble proteins were fractionated by SDS-PAGE and blots were probed with anti-pSer38vim and β-actin antibodies. (B) RbCF2, RbCF5 and RbCF8 cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.g001" target="_blank">Fig 1</a>, plated for 30 min and then treated in presence or absence of WFA or Epox for 30 min. Equal amount of soluble proteins were fractionated by SDS-PAGE and blots were probed with anti-pSer38vim and β-actin antibodies. Asterisk marks the 67-kDa high molecular weight pSer38vim hyperphosphorylated species, and the arrow and arrowheads represent the 57 kDa and 61 kDa pSer38vim bands, respectively. (C) Western blots of RbCF2, RbCF5 and RbCF8 cells re-probed with anti-ubiquitin antibody. (D) Western blots comparing sVim expression in RbCF2 and RbCF8 cells cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133399#pone.0133399.g001" target="_blank">Fig 1</a> and treated with Epox or WFA as above. The arrow indicates the major vimentin 57 kDa species. (E) Western blots of RbCF8 cells treated with WFA in suspension culture. Serum-starved cells were trypsinized and transferred into non-adhesive petri dishes in 10% serum-containing medium in presence or absence of WFA for 1 h. Equal amount of soluble proteins were fractionated by SDS-PAGE and blots were probed with anti-pSer38vim and β-actin antibodies. The arrow points to the 57 kDa species, arrowhead to 61 kDa species and asterisk to the 67 kDa species.</p

Identification of RbTCF cells <i>in vivo</i> and <i>in vitro</i>.

<p>Immunohistochemistry analysis of cell marker vimentin in rabbit Tenon's capsule tissue (B, <i>red</i>) and in isolated RbTCFs (A, <i>green</i>). Scale bars: 100 µm <i>in vitro</i> (A), 50 µm <i>in vivo</i> (B).</p

WFA prevents myofibroblast transformation and collagen gel contraction in RbTCF cells.

<p>Immunohistochemical analysis of vimentin (<i>green</i>) and α-SMA (<i>red</i>) in RbTCF cells treated with TGF-β1 (2 ng/ml) for 3 days in presence and absence of WFA. (A) TGF-β1 treatment induces formation of spheroid-like agglomerates (upper panels, 10× magnification) with sprouting extensions positive for vimentin (<i>green</i>) and α-SMA (<i>red</i>) (lower panels, 30× magnification). (B–C) WFA causes a dose-dependent downregulation of both vimentin (<i>green</i>) and α-SMA (<i>red</i>) expression. Upon treatment with 500 nM WFA, cells form spheroids poorly (C, insert panels) and those that do form spheroids have shorter and thinner extensions (C, <i>arrowheads</i>). Scale bars: 70 µm at 10× magnification; 30 µm at 30× magnification. (D) Western blot analysis of soluble vimentin and α-SMA expression in RbTCF cells (left panel) and RbCF cells (right panel). (E) Densitometric quantification of vimentin (<i>black and orange</i>) and α-SMA (<i>grey and blue</i>) in RbTCF cells and RbCF cells, respectively, normalized to GAPDH using ImageJ software. (F) Representative images of polymerized collagen gels containing RbTCF cells (n = 6 gels per treatment group) treated with TGF-β1 (2 ng/ml) in presence and absence of different doses of WFA for 3 days (10× magnification). (G) Quantification of percentage of gel contraction normalized to the initial size of the gel. Data are the mean of 2 independent experiments; Dotted circles represent the well's area. *P<0.05 TGF-β vs WFA treated cells.</p